Detection of rpoB sequences of mycobacterium tuberculosis

ABSTRACT

A method of detecting rpoB sequences of  Mycobacterium tuberculosis  present in a biological sample that includes steps of amplifying the  M. Tuberculosis  rpoB sequence in vitro in a nucleic acid amplification mixture that includes specific disclosed primer sequences, and detecting the amplified sequences by using probes that provide information by their specific hybridization to portions of the amplified nucleic acid is disclosed. Compositions for amplifying and detecting in vitro the rpoB sequences of  M. Tuberculosis  in a sample are disclosed.

RELATED APPLICATIONS

This application is a divisional of application Ser. No. 10/245,988,filed Sep. 18, 2002 now U.S. Pat. No. 7,094,542, which claims thebenefit under 35 U.S.C. 119(e) of provisional application No.60/323,485, filed Sep. 18, 2001, both of which are incorporated byreference.

FIELD OF THE INVENTION

This invention relates to in vitro diagnostic detection of pathogenicbacteria, and specifically relates to compositions and assays fordetecting nucleic acid sequences associated with rifampin resistance ofMycobacterium Tuberculosis by using in vitro nucleic acid amplificationof the rpoB gene and detection of amplified products.

BACKGROUND OF THE INVENTION

Rifampin (RIF), an antibiotic synthesized from rifamycin B, is a keycomponent of drug therapy against Mycobacterium tuberculosis. Rifampinhas a unique site of action on the beta subunit of prokaryotic RNApolymerase. In Escherichia coli, missense mutations and short deletionsin the central region of the RNA polymerase subunit gene (rpoB) resultin strains resistant to rifampin (Lisityn et al., 1984, Mol. Gen. Genet.196: 173-174). Similarly, in M. Tuberculosis a wide variety of mutationsin the rpoB gene have been identified that confer rifampin resistance(Telenti et al., 1993, Lancet 341: 647-650). More than 90% ofrifampin-resistant M. Tuberculosis isolates are also resistant toisoniazid, and, therefore, rifampin resistance is a valuable surrogatemarker for multiple drug resistance. Thus, there is a need for teststhat can detect rapidly the genetic basis for rifampin resistance fordiagnosis that leads to appropriate treatment of infected individuals.

Early detection of drug resistance in clinical M. Tuberculosis isolatesis crucial for appropriate treatment and to prevent the spread ofresistant strains. Conventional methods of detecting drug-resistance bygrowth of M. Tuberculosis on solid media, and more recent methods thatrely on growth in liquid media have provided susceptibility results in 3days to over 4 weeks (Rusch-Gerdes et al., 1999, J. Clin. Microbiol 37:45-48).

Genetic techniques that rely on the polymerase chain reaction (PCR) havebeen devised to detect rifampin resistance. Such techniques includedirect sequencing of PCR products, single strand conformationpolymorphism analysis, heteroduplexing and dideoxy fingerprinting(Telenti et al., 1993, Lancet 342: 841-844; Williams et al., 1994,Antimicrobial Agents Chemotherapy 38: 2380-2386; De Beenhouwer, 1995,Tubercle and lung disease 76: 425-430). Other assays and reagents fordetecting resistance to rifampin in M. Tuberculosis isolates oridentifying Mycobacteria species using rpoB gene have been previouslydisclosed, for example, in U.S. Pat. No. 5,643,723 (Persing et al.),U.S. Pat. No. 5,851,763 (Heym et al.), U.S. Pat. No. 6,228,575 (Gingeraset al.), and U.S. Pat. No. 6,242,584 (Kook et al.),

The present invention provides compositions and simple diagnosticmethods to detect rifampin resistance in M. Tuberculosis that may bepresent in a clinical sample.

SUMMARY OF THE INVENTION

According to one aspect of the invention, there is provided a method ofdetecting rpoB sequences of Mycobacterium tuberculosis present in abiological sample. The method includes the steps of providing abiological sample containing nucleic acid from M. Tuberculosiscomprising a rpoB sequence; amplifying the rpoB sequence in an in vitronucleic acid amplification reaction mixture comprising at least onepolymerase activity, and at least two primers selected from the groupconsisting of SEQ ID NO:2 with SEQ ID NO: 3, SEQ ID NO:2 with SEQ IDNO:8, and SEQ ID NO:10 with SEQ ID NO:11, to produce amplified nucleicacid containing a rpoB sequence; optionally fragmenting the amplifiednucleic acid; hybridizing the amplified nucleic acid to at least onedetection probe that hybridizes specifically to M. Tuberculosissequences; and detecting the amplified nucleic acid hybridized to atleast one detection probe by detecting a label associated with theamplified nucleic acid. In one embodiment, before the amplifying step,the method also includes the steps of adding to the biological sample atleast one capture oligomer comprising a sequence contained in SEQ IDNO:5, SEQ ID NO:6 or SEQ ID NO:9 that specifically hybridizes to a M.Tuberculosis sequence, and an immobilized nucleic acid that hybridizesto a 3′ tail sequence of the capture oligomer; hybridizing the nucleicacid from M. Tuberculosis, the capture oligomer, and the immobilizednucleic acid to produce a hybridization complex comprising the nucleicacid from M. Tuberculosis, the capture oligonucleotide, and theimmobilized nucleic acid; and separating the hybridization complex fromother components of the biological sample. In another embodiment, thedetecting step uses at least one detection probe that hybridizesspecifically to a rpoB sequence. Another embodiment uses at least onedetection probe consisting of the sequence of SEQ ID NO:4 or SEQ IDNO:12. Another embodiment, in the detecting step, uses a plurality ofdetection probes in a DNA probe array, wherein at least one detectionprobe in the array hybridizes specifically to a rpoB sequence. In oneembodiment, the amplifying step uses primers of SEQ ID NO:2 with SEQ IDNO:3, and a helper oligomer consisting of SEQ ID NO:1. In anotherembodiment, the amplifying step uses primers of SEQ ID NO:2 with SEQ IDNO:8, and a helper oligomer consisting of SEQ ID NO:1. Anotherembodiment uses primers of SEQ ID NO:10 with SEQ ID NO:11. In oneembodiment, the amplifying step uses a transcription-mediatedamplification reaction mixture, whereas in another embodiment, theamplifying step uses a polymerase chain reaction amplification reactionmixture. In one embodiment, the optional fragmenting step includeschemically fragmenting the amplified nucleic acid and fluorescentlylabeling fragments of the amplified nucleic acid.

Other aspects of the invention include various compositions fordetecting a rpoB sequence of M. Tuberculosis. One composition includesoligonucleotides consisting of SEQ ID NO:2, SEQ ID NO:3, and SEQ IDNO:4. Another composition includes oligonucleotides consisting of SEQ IDNO:2, SEQ ID NO:3, and a DNA probe array wherein at least one detectionprobe in the array hybridizes specifically to a rpoB sequence. Anotherembodiment is a composition that includes oligonucleotides consisting ofSEQ ID NO:2, SEQ ID NO:8, and SEQ ID NO:4. Yet another embodiment is acomposition that includes oligonucleotides consisting of SEQ ID NO:2,SEQ ID NO:8, and a DNA probe array wherein at least one detection probein the array hybridizes specifically to a rpoB sequence. Anothercomposition of the invention includes oligonucleotides consisting of SEQID NO:10, SEQ ID NO:11, and SEQ ID NO:12.

Another aspect of the invention is a kit that includes at least twooligonucleotides having sequences selected from the group consisting ofSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:11, and SEQ ID NO:12.

DETAILED DESCRIPTION

The present invention includes methods of detecting rpoB sequences forMycobacterium tuberculosis present in biological samples derived fromhumans, preferably in processed sputum samples. The present inventionalso includes compositions that include nucleic acid capture oligomersthat specifically hybridize to M. Tuberculosis sequences present in abiological sample, thereby providing a means for capturing the targetsequence from the sample components, nucleic acid amplificationoligomers (or primers) that specifically amplify selected portions ofthe rpoB DNA sequences, and nucleic acid probe oligomers (or detectionprobes) for detecting such amplified sequences.

The nucleic acid sequences of this invention are useful for capturing,amplifying and detecting mutations of rpoB gene for M. Tuberculosispresent in a biological sample. The methods of the present invention areimportant for diagnosis of a drug resistance phenotype of M.Tuberculosis by giving the clinician information useful in determiningappropriate treatment of the M. Tuberculosis infected patient.

The following definitions are provided to aid in understanding thedescribed invention.

By “biological sample” is meant any tissue or material derived from aliving or dead human which may contain M. Tuberculosis nucleic acid.Samples include, for example, sputum, respiratory tissue or exudates,peripheral blood, plasma or serum, cervical swab samples, biopsy tissue,gastrointestinal tissue, urine, feces, semen or other body fluids,tissues or materials. Samples also include bacterial cultures (fromliquid or solid media) and environmental samples. A biological samplemay be treated to physically disrupt tissue or cell structure, thusreleasing intracellular components into a solution which may containenzymes, buffers, salts, detergents and the like which are used toprepare the sample for analysis.

By “nucleic acid” is meant a multimeric compound comprising nucleosidesor nucleoside analogs which have nitrogenous heterocyclic bases, or baseanalogs, where the nucleosides are covalently linked via a backbonestructure to form a polynucleotide. Conventional RNA, DNA, and analogsof RNA and DNA are included in this term. A nucleic acid backbone maycomprise a variety of known linkages known, including one or more ofsugar-phosphodiester linkages, peptide-nucleic acid bonds (“peptidenucleic acids”; PCT No. WO 95/32305 (Hydig-Hielsen et al.)),phosphorothioate linkages, methylphosphonate linkages or combinations ofknown linkages. Sugar moieties of the nucleic acid may be ribose ordeoxyribose, or similar compounds having known substitutions, e.g., 2′methoxy and/or 2′ halide substitutions. Nitrogenous bases may beconventional bases (A, G, C, T, U), known base analogs (e.g., inosine;see The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed.,11^(th) ed., 1992), or known derivatives of purine or pyrimidine bases(PCT No. WO 93/13121 (Cook)) and “abasic” residues in which the backboneincludes no nitrogenous base for one or more residues (U.S. Pat. No.5,585,481 (Arnold et al.)). A nucleic acid may comprise onlyconventional sugars, bases and linkages, as found in RNA and DNA, or mayinclude both conventional components and substitutions (e.g.,conventional bases linked via a methoxy backbone, or a nucleic acidincluding conventional bases and one or more analogs).

By “oligonucleotide” or “oligomer” is meant a nucleic acid havinggenerally less than 1,000 residues, including those in a size rangehaving a lower limit of about 2 to 5 nucleotide residues and an upperlimit of about 500 to 900 nucleotide residues. Oligomers may be in asize range having a lower limit of about 5 to about 15 residues and anupper limit of about 50 to 600 residues; and preferably, in a size rangehaving a lower limit of about 10 residues and an upper limit of about100 residues. Oligomers can be purified from natural sources, butgenerally are synthesized in vitro using well-known methods.

By “amplification oligonucleotide” or “amplification oligomer” is meantan oligonucleotide or oligomer that hybridizes to a target nucleic acid,or its complement, and participates in an in vitro nucleic acidamplification reaction. These may be called “primers” because theyinitiate polymerization from a template by enzymatic activity that addsnucleotide monomers at their 3′ ends. An amplification oligonucleotidegenerally contains at least 10 to 12 contiguous bases that arecomplementary to a region of the target nucleic acid sequence (or itscomplementary strand). The contiguous bases are preferably at least 80%,more preferably at least 90% complementary to the sequence to which theamplification oligonucleotide binds. An amplification oligonucleotide ispreferably about 10 to about 60 bases long and may include modifiednucleotides, base analogs or additional functional sequences, such as a5′ promoter sequence recognized by an RNA polymerase (such amplificationoligonucleotides may be called “promoter primers”).

Those skilled in the art will appreciate that any oligomer that canfunction as a primer can be modified to include a 5′ promoter sequence,and thus function as a promoter primer. Similarly, any promoter primercan serve as a primer, independent of its promoter sequence.

By “amplification” is meant an in vitro procedure for obtaining multiplecopies of a target nucleic acid sequence or its complement or fragmentsthereof. In vitro amplification refers to production of an amplifiednucleic acid that may contain less than the complete target regionsequence or its complement. Known amplification methods include, forexample, transcription-mediated amplification (TMA), replicase-mediatedamplification, polymerase chain reaction (PCR) amplification, ligasechain reaction (LCR) amplification and strand-displacement amplification(SDA). Replicase-mediated amplification uses self-replicating RNAmolecules, and a replicase such as Q Beta-replicase (U.S. Pat. No.4,786,600 (Kramer et al.) and U.S. Pat. No. 5,112,734 (Lizardi et al.)).PCR amplification uses thermal cycling with a DNA polymerase and,usually, two or more primers to synthesize multiple copies of twocomplementary DNA strands (Mullis et al., 1987, Methods in Enzymology155: 335-350; U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159 (Mulliset al.)). LCR amplification uses at least four separate oligonucleotidesto amplify a target and its complementary strand by using multiplecycles of hybridization, ligation, and denaturation (EP Pat. No. 0320308(Wang et al.)). SDA uses a primer that contains a recognition site for arestriction endonuclease which will nick one strand of a hemimodifiedDNA duplex that includes the target sequence, followed by a series ofprimer extension and strand displacement steps to amplify DNA (U.S. Pat.No. 5,422,252 (Walker et al.)). Transcription-mediated amplification(TMA) is used in preferred embodiments of the present invention. Thoseskilled in the art will understand that the oligonucleotide sequences ofthe present invention may be readily used in any in vitro amplificationmethod based on primer extension.

By “transcription-mediated amplification” or “transcription-associatedamplification” is meant nucleic acid amplification that uses an RNApolymerase to produce multiple RNA transcripts from a nucleic acidtemplate. TMA generally uses an RNA polymerase activity, a DNApolymerase activity, deoxyribonucleoside triphosphates, ribonucleosidetriphosphates, and a promoter primer and a second primer, and optionallymay include one or more additional oligonucleotides (sometimes referredto as “helper” or “displacer” oligonucleotides). These amplificationmethods are well known in the art, as described in detail elsewhere(U.S. Pat. Nos. 5,399,491 and 5,554,516 (Kacian et al.), U.S. Pat. No.5,786,183 (Ryder et al.), PCT No. WO 93/22461 (Kacian et al.); U.S. Pat.No. 5,437,990 (Burg et al.); PCT Nos. WO 88/01302 and WO 88/10315(Gingeras et al.); U.S. Pat. No. 5,130,238 (Malek et al.); U.S. Pat.Nos. 4,868,105 and 5,124,246 (Urdea et al.); PCT No. WO 94/03472(McDonough et al.); and PCT No. WO 95/03430 (Ryder et al.)). PreferredTMA methods have been disclosed in U.S. Pat. Nos. 5,399,491, 5,554,516and 5,786,183, and PCT No. WO 93/22461.

By “probe” is meant a nucleic acid oligomer that hybridizes specificallyto a target sequence in a nucleic acid or its complement, preferably inan amplified nucleic acid, under conditions that promote hybridization,thereby allowing detection of the target or amplified nucleic acid.Detection may either be direct (i.e., resulting from a probe hybridizingdirectly to the target sequence or amplified nucleic acid) or indirect(i.e., resulting from a probe hybridizing to an intermediate molecularstructure that links the probe to the target sequence or amplifiednucleic acid). A probe's “target” generally refers to a sequence in(i.e., a subset of) a larger nucleic acid sequence that hybridizesspecifically to at least a portion of the probe sequence by standardhydrogen bonding (base pairing). Sequences that are “sufficientlycomplementary” allow stable hybridization of a probe oligomer to atarget sequence, even if the two sequences are not completelycomplementary. A probe may be labeled or unlabeled, depending on thedetection method used, which methods are well known in the art.

By “sufficiently complementary” is meant a contiguous nucleic acid basesequence that hybridizes to another base sequence by hydrogen bondingbetween a series of complementary bases under hybridization conditions.Sequences may be complementary at each position in a sequence usingstandard base pairing (i.e., G:C, A:T or A:U pairing) or may contain oneor more residues that are not complementary by standard hydrogen bonding(including abasic residues), but in which the entire base sequence iscapable of specifically hybridizing with another base sequence inappropriate hybridization conditions. Contiguous bases are preferably atleast about 80%, more preferably at least about 90% complementary to asequence to which an oligomer specifically hybridizes. Appropriatehybridization conditions are well known to those skilled in the art, canbe predicted readily based on sequence composition and conditions, orcan be determined empirically by using routine testing (Sambrook et al.,Molecular Cloning, A Laboratory Manual, 2^(nd) ed. (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989) at §§ 1.90-1.91,7.37-7.57, 9.47-9.51 and 11.47-11.57, particularly at §§ 9.50-9.51,11.12-11.13, 11.45-11.47 and 11.55-11.57).

By “capture oligonucleotide” or “capture oligomer” or “capture probe” ismeant at least one nucleic acid oligomer that provides means forspecifically joining a target sequence and an immobilized oligomer basedon base pair hybridization (U.S. Patent No. 6,110,678 (Weisburg etal.)). Generally, a capture oligomer includes two binding regions: atarget-specific binding region and an immobilized probe-specific bindingregion.

By “immobilized probe” or “immobilized oligomer” is meant a nucleic acidthat joins, directly or indirectly, a capture oligomer to a solidsupport. An immobilized probe is an oligonucleotide joined to a solidsupport provides a means for separating a bound target sequence fromother sample components. Suitable solid supports include matrices andparticles in solution, made of any known material (e.g., nitrocellulose,nylon, glass, polyacrylate, mixed polymers, polystyrene, silanepolypropylene and metal particles, preferably paramagnetic particles).Preferred supports are monodisperse paramagnetic spheres (uniform insize +about 5%), to which an immobilized probe is stably joined directly(e.g., via direct covalent linkage, chelation, or ionic interaction), orindirectly (e.g., via hybridization with one or more linkers), thuspermitting hybridization to another nucleic acid in solution.

By “separating” or “purifying” is meant that one or more components ofthe biological sample are removed from other sample components. Samplecomponents generally are an aqueous solution that includes nucleic acidsand other materials (e.g., proteins, carbohydrates, lipids and/ornucleic acids). A separating or purifying step removes at least about70%, preferably at least about 90%, and more preferably at least about95% of the other sample components.

By “label” is meant a molecular moiety or compound that can be detectedor can lead to a detectable response. A label is joined, directly orindirectly, to a nucleic acid probe or to a nucleic acid to be detected(e.g., an amplified nucleic acid). Direct labeling can occur throughbonds or interactions that link the label to the probe (e.g., viacovalent bonds or non-covalent interactions). Indirect labeling canoccur through use of a bridging moiety or linker, such as additionaloligonucleotide(s), which is either directly or indirectly labeled.Bridging moieties can be used to amplify detectable signal. Labels canbe any known detectable moiety (e.g., radionuclide, ligand, such asbiotin or avidin, enzyme, enzyme substrate, reactive group, chromophore,e.g., dye or colored particle, luminescent compound, includingbioluminescent, phosphorescent, chemiluminescent and fluorescentcompounds). Preferably, the label on a labeled probe is detectable in ahomogeneous assay system (i.e., in a mixture, bound labeled probeexhibits a detectable change compared to unbound labeled probe).Preferred chemiluminescent labels and their use in homogenous detectionassays have been described in detail (U.S. Pat. No. 5,283,174 (ArnoldJr., et al.), U.S. Pat. No. 5,656,207 (Woodhead et al.), U.S. Pat. No.5,658,737 (Nelson et al.) and U.S. Pat. No. 5,639,604 (Arnold Jr., etal.)). Such labels include acridinium ester (“AE”) compounds, e.g.,standard AE or its derivatives. A homogeneous detectable label has theadvantage of being detectable without physically separating hybridizedfrom unhybridized label or labeled probe. Methods of attaching labels tonucleic acids and detecting labels are well known in the art (Sambrooket al., Molecular Cloning, A Laboratory Manual, 2^(nd) ed. (Cold SpringHarbor Laboratory Press, Cold Spring Habor, N.Y., 1989), Chapter 10;U.S. Pat. No. 5,731,148 (Becker et al.), U.S. Pat. No. 5,658,737 (Nelsonet al.), U.S. Pat. No. 5,656,207 (Woodhead et al.), U.S. Pat. No.5,547,842 (Hogan et al.), U.S. Pat. No. 5,283,174 (Arnold Jr., et al.)and U.S. Pat. No. 4,581,333 (Kourilsky at al.)).

By “DNA probe array”, is meant a solid support on which are immobilizedat least 2, and preferably 10 or more, different captureoligonucleotide. Examples of such DNA probe arrays are well known in theart (Ramsay, 1998, Nature Biotech. 16: 40-44; Cheng et al., 1996, Molec.diagnosis 1 (3): 183-200; Livache et al., 1994, Nucl. Acids Res. 22(15): 2915-2921; Cheng et al., 1998, Nature Biotech. 16: 541-546; U.S.Pat. No. 4,981,783 (Augenlicht), U.S. Pat. No. 5,700,637 (Southern),U.S. Pat. Nos. 5,445,934 and 5,744,305 (Fodor), and U.S. Pat. No.5,807,522 (Brown)).

By “consisting essentially of” is meant that additional component(s),composition(s) or method step(s) that do not materially change the basicand novel characteristics of the invention may be included in thecompositions, kits or methods of the present invention. Suchcharacteristics include the ability to detect rpoB sequences of M.Tuberculosis in a biological sample at about 20 to 200 or more copiesper sample. Any component, composition, or method step that has amaterial effect on the invention's basic characteristics would falloutside of this term.

Unless defined otherwise, all scientific and technical terms used hereinhave the same meaning as commonly understood by those skilled in therelevant art. General definitions are provided, for example, inDictionary of Microbiology and Molecular Biology, 2^(nd) ed. (Singletonet al., 1994, John Wiley & Sons, New York, N.Y.) or The Harper CollinsDictionary of Biology (Hale & Marham, 1991, Harper Perennial, New York,N.Y.). Unless mentioned otherwise, the techniques employed orcontemplated herein are well known standard methods in the art.

The present invention includes compositions (nucleic acid captureoligomers, amplification oligomers and probes) and methods for detectingM. Tuberculosis rpoB nucleic acid in a human biological sample. Toselect appropriate DNA sequences for such use, known rpoB sequences fromdifferent Mycobacterium isolates, including known mutations, availablefrom publicly accessible databases (e.g., GenBank) were aligned bymatching regions of the same or similar sequences and compared usingwell known molecular biology techniques. Although use of algorithms mayfacilitate sequence comparisons, those skilled in the art can readilyperform such comparisons manually and visually. Generally, sequenceportions containing relatively few variants between the comparedsequences were chosen as a basis for designing synthetic oligomerssuitable for use in capture and amplification of M. Tuberculosis nucleicacid sequences in the rpoB region and detection of amplified sequences.Other considerations in designing oligomers included the relative GCcontent which affects T_(m) of the sequence and the relative absence ofpredicted secondary structure within a sequence, all well known in theart. Based on these analyses, the oligomers having sequences of SEQ IDNO:1 to SEQ ID NO:6 and SEQ ID NO:8 to SEQ ID NO:12 were designed andsynthesized.

Target capture may be included to increase the concentration or purityof the target nucleic acid before in vitro amplification. Preferably,target capture involves a relatively simple method of hybridizing andisolating the target nucleic acid, as described in detail elsewhere(U.S. Pat. No. 6,110,678 and PCT No. WO 98/50583 (Weisburg et al.)).Briefly, a sample that potentially contains M. Tuberculosis DNA iscontacted with a capture oligomer containing a sequence specific forhybridization to M. Tuberculosis DNA, and an immobilized oligonucleotideattached to a solid support that can hybridize to another portion of thecapture oligomer under appropriate hybridization conditions. Followinghybridization of the capture oligomer to the M. Tuberculosis DNA andthen to the immobilized oligonucleotide, the hybridization complexattached to the solid support is separated from other sample components.Then, the M. Tuberculosis target nucleic acid linked to the solidsupport is washed and rpoB sequences are amplified in an in vitroamplification reaction.

The capture oligomer sequence includes a 5′ target-binding sequence thatbinds specifically to the M. Tuberculosis DNA target sequence, and a 3′tail sequence (e.g., poly-dA) that binds to the complementaryimmobilized sequence (e.g., poly-dT) on the solid support. A captureoligomer may use any backbone to link the base sequence, includingstandard deoxyribose-phosphate linkages and O-methoxy linkages.Preferred capture oligomers have sequences of:GGCCACCATCGMTATCTGGTCCGCTTGCACTTT(A)₃₀ (SEQ ID NO:5),CATGTCGCGGATGGAGCGGGTGGTC(A)₃₀ (SEQ ID NO:6), andCATCGMTATCTGGTCCGCTTGCAC(A)₃₀ (SEQ ID NO:9).

Amplifying the captured rpoB region can be accomplished using a varietyof known nucleic acid amplification reactions, but preferably uses atranscription-mediated amplification (TMA). Using such an in vitroamplification method, many strands of nucleic acid are produced from asingle copy of target nucleic acid, thus permitting detection of thetarget by specifically binding the amplified rpoB sequences to one ormore detecting probes. TMA has been described in detail previously (U.S.Pat. Nos. 5,399,491 and 5,554,516 (Kacian et al.)). Briefly, thisamplification method uses two types of primers (one being a “promoterprimer” that contains a promoter sequence for an RNA polymerase), twoenzymes (a reverse transcriptase and an RNA polymerase), substrates(deoxyribonucleoside triphosphates, ribonucleoside triphosphates) andappropriate salts and buffers in solution to produce multiple RNAtranscripts from a nucleic acid template. First, a promoter primerhybridizes specifically to its target nucleic acid sequence and reversetranscriptase creates a first strand cDNA by extension from the 3′ endof the promoter primer. The cDNA becomes available for hybridizationwith the second primer by enzymatic activity that degrades thecomplementary strand (e.g., RNase H activity of the reversetranscriptase) or by localized or complete denaturaton of the duplex. Asecond primer binds to the cDNA and a new strand of DNA is synthesizedfrom the 3′ end of the second primer using reverse transcriptase tocreate a double-stranded DNA having a functional promoter sequence atone end. RNA polymerase binds to the double-stranded promoter sequenceand transcription produces multiple transcripts or “amplicons.”Amplicons are used in further steps in the process, each serving as atemplate for a new round of replication as described above, thusgenerating large amounts of single-stranded amplified nucleic acid in asubstantially isothermal process. For example, about 100 to about 3,000copies of RNA transcripts are synthesized from a single template.

Primer sequences (e.g., SEQ ID NO: 1 to SEQ ID NO: 3 and SEQ ID NO:8,SEQ ID NO:10 and SEQ ID NO:11) bind specifically to an rpoB targetsequence or its complement, but such primer sequences may containsequences that do not bind to the target sequence or its complement. Forexample, promoter primers (e.g., SEQ ID NO:2, SEQ ID NO:10) may includea T7 RNA polymerase promoter sequence (SEQ ID NO:7) as a 5′ portion ofthe sequence.

Embodiments of the present invention are described in the examples thatfollow. Briefly, the methods include the steps of providing a biologicalsample that potentially contains the target M. Tuberculosis rpoB gene,target capture of DNA containing an rpoB sequence, in vitro nucleic acidamplification and detection of the amplified nucleic acid products todetermine if an rpoB mutation is present in the amplified nucleic acid.If an rpoB mutation is detected, it indicates that the sample containeda RIF-resistant M. Tuberculosis. In preferred embodiments that usetranscription-mediated amplification (TMA), the amplification mixtureincludes the captured target DNA, at least one T7 promoter primer thatincludes a target-specific sequence and a T7 promoter sequence, at leastone second primer that hybridizes specifically to a first strand cDNAmade from the target using the T7 promoter primer, and substrates andcofactors for enzymatic polymerization by reverse transcriptase and T7RNA polymerase. The captured target does not have to be separated fromthe solid support for use in the TMA reaction. The functional T7promoter sequence combined with T7 RNA polymerase produces multipletranscripts which can be detected using any of a variety of knownmethods, including hybridizing specifically the amplified products, orportions thereof, to one or more complementary probe sequences. In someembodiments, a labeled probe is used to detect the amplified products,whereas in other embodiments, the amplified products are labeled andhybridized to immobilized probes, preferably to an array of many probes.The hybridization complex of the probe and amplified product isdetected. When an array of different probes is used, the pattern ofhybridization on the array indicates the sequence of the amplified rpoBgene, which provides information on whether a rpoB mutation of M.Tuberculosis is present in the sample assayed.

Typical assay conditions described as follows.

Sample preparation. A sample (e.g., 0.5 ml of sputum sediment orbacterial culture) was mixed with an equal volume of a 2× lysis buffer(e.g., 20 mM HEPES, 0.5% (w/v) lithium lauryl sulfate (LLS), pH 8). Torelease nucleic acids from the bacteria, the mixture was vortexed in thepresence of glass beads, or sonicated for 15 min, and then the mixturewas heated at 95° C. for 15 min. For positive control reactions, anequal volume of water or buffer containing a known amount of M.Tuberculosis genomic DNA (gDNA) was used in place of the sputum sedimentor bacterial culture. The gDNA was prepared using a combination ofstandard methods that have been described in detail previously. Briefly,cells were grown in broth to late log phase and treated 18 hr with 1mg/ml ampicillin and 0.1 mg/ml D-Cycloserine (Crawford et al., 1979,Infect. Immun. 24: 979-81). Then, cells were collected and lysed withSDS and treated with Proteinase K to release DNA into an aqueoussolution which was extracted twice with sodium perchlorate and aphenol/chloroform mixture, and DNA was spooled out of the solution afteradding about two volumes of ethanol (Maniatis et al., Molecular Cloning,A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1982), pp. 280-81 and 458-59; and Marmur, 1961, J. Mol.Biol. 3: 209-18).

Target capture. Generally, lysate prepared from a sample was used in thetarget capture step (U.S. Pat. No. 6,110,678 (Weisburg et al.)). Tocapture the target M. Tuberculosis DNA, the mixture included 250 μl ofprepared sample lysate, 250 μl of a target capture solution containing 3pmole of SEQ ID NO:5 and 3 pmole of SEQ ID NO:6 or 3 pmol of SEQ IDNO:9, and 40 μg of paramagnetic particles (0.7-1.05μ, Seradyn,Indianapolis, Ind.) with attached immobilized poly-dT₁₄ oligomers (Lund,et al., 1988, Nuc. Acids Res. 16: 10861-80). The target capture mixturewas heated at 60° C. for about 20 min and then cooled to roomtemperature. A magnetic field was applied for 5 min to attract themagnetic particles with the attached complex containing the target DNAto a location on the reaction container (substantially as described inU.S. Pat. No. 4,895,650 (Wang)). Particles with attached tohybridization complexes were washed twice with 1 ml of a washing buffer(10 mM HEPES, 6.5 mM NaOH, 1 mM EDTA, 150 mM NaCl, 0.1% (w/v) sodiumlauryl sulfate) by resuspending the particles in the washing buffer andthen repeating the magnetic separation step.

Amplification. Transcription mediated amplification was performedsubstantially as described previously (U.S. Pat. Nos. 5,399,491 and5,554,516 (Kacian et al.)). Washed particles from the target capturestep were suspended in 75 μl of amplification reagent solution (0.08 mMrUTP, 1.3 mM rATP, 4 mM rCTP, 6 mM rGTP, 1.3 mM each dNTP, 66 mM Tris,17.3 mM MgCl₂). The relatively low concentration of rUTP is importantfor amplification efficiency of the rpoB target DNA. At least twoamplification oligomers were included in the amplification reaction,i.e., at least one promoter primer and a second primer, usually at 0.08μM final concentration. (Amplification oligomers may also include helperor displacer oligomers and may be hybridized to the target before otheramplification reagents are added to the mixture.) The reaction mixturewas covered with a layer (200 μl) of inert oil to prevent evaporationand incubated at 42° C. for 5 min. Then 25 μl of enzyme reagent wasadded (about 1750 U of MMLV reverse transcriptase and 400 U of T7 RNApolymerase per reaction, in a buffer containing 50 mM HEPES, 1 mM EDTA,10% (v/v) t-octylphenoxypolyethoxyethanol (TRITON™ X-100), 120 mM KCl,20% (v/v) glycerol). (One unit of MMLV reverse transcriptaseincorporates 1 nmol of dTTP in 10 min at 37° C. using a polyA templateprimed with 200-400 μM oligo(dT); and one unit of T7 RNA polymeraseincorporates 1 nmol of ATP into RNA in 1 hr at 37° C. using a DNAtemplate containing a T7 promoter sequence.) After mixing gently, thereaction was incubated at 42° C. for 1 hr. Negative controls consistedof all of the same reagents but substituting an equal volume of water orbuffer that contained no target.

Detection. In some cases, amplified M. Tuberculosis sequences weredetected using an acridinium ester (AE)-labeled probe (e.g.,5′-GTTGTTCTGGTCCATGAA (SEQ ID NO:4)) which was detected bychemiluminescence in a luminometer (e.g., LEADER™ luminometer, Gen-ProbeIncorporated, San Diego, Calif.) and signal is expressed in relativelight units (RLU) substantially as described previously (U.S. Pat. No.5,658,737 (Nelson et al.) at column 25, lines 27-46; Nelson et al.,1996, Biochem. 35: 8429-8438, at 8432). Generally, the average (mean) ofdetected RLU for replicate assays are reported. In a preferredembodiment, the labeled detection probe has the base sequence of SEQ IDNO:4 linked by a 2′-O-methoxy backbone.

In other cases, the amplified sequences were detected on an immobilizedarray of DNA probes specific for detection of M. Tuberculosis rpoBsequences, as described in detail previously (Troesch et al., 1999, J.Clin. Microbiol. 37: 49-55). Amplicons generated by the amplificationreaction were labeled with a fluorescent label before hybridization tothe array using methods substantially as described in detail elsewhere(PCT Nos. WO 99/65926 and WO 01/44507 (Laayoun et al.)). Briefly 50 μlof amplicons were mixed with 30 mM MnCl₂, 30 mM imidazole, 2mM of5-(bromomethyl)fluorescein and water (150 μl final volume). After a 30min incubation at 65° C., free label was eliminated by columnchromatography (e.g., using a 6S QIAVAC® column, Qiagen GmbH, accordingto the manufacturer's instructions).

Hybridization of the probe arrays was performed with the GENECHIP™Fluidics Station (Affymetrix, Santa Clara, Calif.) substantially aspreviously described (Troesch et al., 1999, J. Clin. Microbiol. 37:49-55). An additional step, antibody staining, allows signalamplification as described elsewhere (PCT No. WO 01/44506 (Laayoun etal.)). Briefly, after hybridization was performed on the DNACHIP™ usingthe protocol of Troesch et al., the DNACHIP™ was flushed and a secondstep of staining was performed using staining solution containing 300 μlof 2 M MES, 2.4 μl of bovine serum albumin (BSA), 6 μl of normal goatIgG, 1.2 μl of anti-fluorescein antibody, and water (600 μl finalvolume). Anti-fluorescein, rabbit IgG fraction, biotin-XX conjugate,were supplied by Molecular Probes (Eugene, Oreg.); acetylated BSAsolution was supplied by GibcoBRL Life Technologies, (Rockville, Md.);and goat IgG (Reagent Grade) was supplied by Sigma Chemical, (St. Louis,Mo.). After a 10 min hybridization, the chip was flushed, washed with awashing buffer containing 6×SSPE, 0.01% polyoxyethylenesorbitan (TWEEN™20), and a third hybridization step was performed, using second stainingsolution of 300 μl of 2M MES, 6 μl of BSA and 6 μl of streptavidin,R-phycoerythrin conjugate, and water (600 μl final volume). Streptavidinand R-phycoerythrin conjugate were supplied by Molecular Probes (Eugene,Oreg.). After a 10 min hybridization, the chip was flushed and washed asdescribed above. The analysis to detect the intensity and pattern offluorescent signals (expressed as relative fluorescence units or RFU) onthe hybridized array was performed on the GENECHIP™ instrumentationsystem (Affymetrix, Santa Clara, Calif.) which comprises a GENECHIP™fluidics station, a GENEARRAY™ scanner (Hewlett-Packard, Palo Alto,Calif.) and GENECHIP™ analysis software (algorithm to determinenucleotide base calling and the nucleic acid sequence of the amplifiednucleic acid). This system generates a report of the rpoB mutationspresent in the amplified nucleic acid sequences applied to the chip.

The following examples demonstrate embodiments of the present invention.

EXAMPLE 1 Sensitivity of Transcription Mediated Amplification Using M.Tuberculosis-Specific Oligonucleotides

This example shows the sensitivity of the amplification oligonucleotidesof the present invention when used in a TMA reaction. Primers weredesigned to amplify M. Tuberculosis specifically and not otherMycobacterium species. Using the target capture and amplificationmethods described above, the efficiencies of transcription mediatedamplification were tested using the following combination ofamplification oligonucleotides: SEQ ID NO:1 (GACCACCCAGGACGTG) as ahelper oligomer, SEQ ID NO: 2(AATTTAATACGACTCACTATAGGGAGACGATCACACCGCAGACGTTG) as a promoter primer,and SEQ ID NO: 3 (GCTCGCGCTCACGTG) as a primer. Target sequences forthis assay were purified gDNA extracted from a lysed bacterial cultureof M. Tuberculosis and provided at 20, 200 or 1000 copies per in vitroamplification reaction. As a negative control, an equal volume of watercontaining no M. Tuberculosis DNA was substituted for the gDNA sample ina separate amplification reaction that was processed as for the positivesamples. Amplification was assessed based on the detectedchemiluminescence (RLU) using a homogeneous detection assay performedsubstantially as described elsewhere in detail (U.S. Pat. No. 5,283,174(Arnold Jr., et al.), U.S. Pat. No. 5,658,737 (Nelson et al.) and U.S.Pat. No. 5,639,604 (Arnold Jr., et al.)). An AE-labeled detection probeof SEQ ID NO:4 (GTTGTTCTGGTCCATGAA) was mixed with unlabeled probe ofthe same sequence (ratio of labeled probe/unlabeled probe was 1/5000) toprovide a signal within the linear range detectable by the LEADER™luminometer (Gen-Probe Incorporated, San Diego, Calif.). Signals of2×10⁴ or greater RLU were considered positive. The RLU results (mean of10 assays for each assay condition) are shown in Table 1.

These results demonstrate that the amplification reaction was sensitiveat as few as 20 copies of gDNA, a level more sensitive than the desiredsensitivity of a clinical smear positive specimen which usually containsat least 1000 bacteria.

TABLE 1 gDNA Copies per Reaction Detected RLU   0 4.17 × 10³ (negativecontrol)  20 3.57 × 10⁴  200 3.96 × 10⁵ 1000 1.31 × 10⁶

In other experiments, the rpoB region was amplified similarly but usinga combination of amplification oligonucleotides of SEQ ID NO:1 as ahelper oligomer, SEQ ID NO: 2 as a promoter primer, and SEQ ID NO:8(CGGCACGCTCACGTG) as primer. The sensitivity of the assay in theseexperiments, as detected by hybridization with an AE-labeled probe, wasat least about 200 copies of target per reaction. In these experiments,the target was provided at 800, 500 and 200 copies per reaction (5reactions for each condition). All reactions gave positive results(7.88×10⁵ to 2.66×10⁶ mean RLU) compared to the negative controls withno M. Tuberculosis DNA in the reaction (which produced 1.93×10³ mean RLUfor two reactions).

EXAMPLE 2 Specificity of Amplification

This example shows the specificity of the amplification oligomers asdemonstrated using a TMA reaction performed using amplificationoligomers and procedures substantially as described in Example 1 andabove. The target sequence for this assay was purified MycobacteriumgDNA extracted from bacteria obtained from the American Type CultureCollection (“ATCC”, Manassas, Va.) or the Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH culture collection (“DSM”,Braunschweig, Germany) and grown in vitro using standard microbiologyprocedures. The species tested included: M. Tuberculosis (ATCC No.27294), M. kansasii (DSM No. 43224), M. avium (ATCC No. 25291), and M.gordonae (ATCC No. 14470). In the amplification reactions, the targetDNA were provided at 200, 10³, 10⁴, 10⁵, and 10⁶ copies per assay. Thenegative control reaction contained no Mycobacterium. DNA and an equalvolume of water was substituted for the sample volume.

For detection of the amplified nucleic acid, the homogeneous detectionassay using a labeled probe as described in Example 1 was used exceptthat undiluted labeled probe was used. The average RLU results (fourassays per species DNA) obtained from these assays are shown in Table 2.For these, a signal of 5×10⁴ RLU or greater was considered positive.

TABLE 2 Copies of Signal Detection Target Results (RLU) Obtained withMycobacterium species per reaction M. tuberculosis M. kansasii M. aviumM. gordonae  0 4 × 10³ 200 4.78 × 10⁶ Not Tested Not Tested Not Tested 10³ Not Tested 1.37 × 10⁴ 1.37 × 10⁴ 1.34 × 10⁴  10⁴ Not Tested 1.68 ×10⁴ 1.67 × 10⁴ 1.51 × 10⁴  10⁵ Not Tested 1.40 × 10⁴ 1.41 × 10⁴ 1.27 ×10⁴

As shown by the results in Table 2, the assay amplified and detected 200copies of M. Tuberculosis DNA in the reactions. For all of the otherMycobacterium species assayed, the results were negative even when muchmore target DNA was used (10³ to 10⁵ copies per reaction). Thus, thespecificity for M. Tuberculosis using these amplification oligomers andprobe was demonstrated by the minimal detectable signal obtained for theother Mycobacterium species even when more DNA was provided.

Using the same amplification procedures as described immediately above,the amplicons were also detected on a DNA probe array. Amplicons arechemically fragmented and fluorescently labeled using proceduressubstantially as previously described (PCT No. WO 01/44507 (Laayoun etal.)). The labeled fragments were detected on the DNA probe array usingthe GENECHIP™ System for detecting M. Tuberculosis sequences asdescribed above.

For each species tested, the results shown in Table 3 present thepercentage of correct base calling that is used to identify apredetermined sequence diagnostic of M. Tuberculosis. For ampliconsobtained from sample DNA from each of the Mycobacterium species listedin Table 3, the relative amount of correct base calling on the M.Tuberculosis-specific DNA probe chip is shown, with the average signalintensity for the detected signal (mean relative fluorescence units orRFU). A result of greater than 85% base calling is considered positiveidentification of the M. Tuberculosis sequence.

The results obtained with this probe array detection system confirmedthat the labeled probe detection results obtained with the homogeneousdetection assay discussed above, and further confirmed that theamplification was specific for M. Tuberculosis.

TABLE 3 M. tuberculosis-specific Species Base Calling % Intensity (RFU)M. tuberculosis 96.7 3146 M. kansasii 9.8 92 M. avium 11.4 60 M.gordonae 13 125

EXAMPLE 3 Detection of Mutant Clones

This example shows the detection of rpoB sequences after target capture,amplification and detection. Detection was done by using labeled probebinding in a homogeneous detection assay and by binding labeledamplicons to a DNA probe array, substantially as described in Example 2.For detection, the same amplification reaction for each clone wasdivided into two parts.

The bacterial rpoB clones to be detected were generated from cloned rpoBsequences contained in a fragment of about 700 bp which was amplifed bythe PCR and ligated into a plasmid vector (pGEM™-T EASY, Promega,Madison, Wis.) as described by Troesch et al. (J. Clin. Microbiol.,1999, 37: 49-55). The insert DNA was sequenced. Clones containing knownmutations served as the M. Tuberculosis target sequence for targetcapture, amplification and detection using the procedures describedabove.

In the results shown in Table 4, a mutation detected in a cloned rpoBsequence is identified by the amino acid substitution (one letter code)and the position of the codon as described by Troesch et al. (id.). Forexample, “Q513L” means that a mutation affected position 513 relative tothe initiation codon, which has a glutamine (Q) in a wild type strainbut has a leucine (L) substitution in this mutant. Column 1 shows theexpected sequence based on the independent sequencing of the clonedinsert, and column 2 shows the results determined by hybridization ofamplicons to the DNA probe array. The percentage of base calling (BC %)and the signal intensity (RFU) observed on the probe array for eachclone are shown in columns 3 and 4, respectively. Column 5 shows therelative amount of M. Tuberculosis amplicons obtained for each clone inone assay, as determined by hybridization to an AE-labeled probe anddetected as relative light units (RLU) as described above. The resultsin Table 4 show that the amplification and detection methods result incorrect identification of different variations that occur in rpoBsequences of M. Tuberculosis mutants.

TABLE 4 DNA probe array results HPA results Expected Observed BC %Intensity (RFU) RLU F505L/L511P/ F505L/L511P/ 100 2,535 7.51 × 10⁶ S531CS531C Q513L Q513L 86.2 1,315 3.50 × 10⁶ H526D H526D 94.3 2,285 9.63 ×10⁶ D516Y D516Y 95.1 1,182 7.94 × 10⁶ H526Y H526Y 97.6 3,108 5.66 × 10⁶L511P L511P 92.7 1,774 4.74 × 10⁶ H526R H526R 96.7 3,422 7.44 × 10⁶

EXAMPLE 4 Detection of M. Tuberculosis in Clinical Specimen

This example shows the sensitivity of primers of the present inventionwhen used in TMA amplification of clinical samples containing wild typeM. Tuberculosis and detection of the amplified RNA. Amplification wasdone substantially as described in Example 1. Amplicons were detectedsubstantially as described in Example 2 on a solid support having anarray of immobilized probes (GENECHIP™) and in a homogeneous detectionassay with a labeled probe.

Positive sediments of M. Tuberculosis (wild type) were obtained fromsputum clinical specimens after digestion and decontamination of thesample. Most specimens received for mycobacterial culture containvarious amounts of organic debris and a variety of contaminating,normal, or transient bacterial flora. A chemical decontamination processkills the contaminants while allowing recovery of the mycobacteria. Thedigestion and decontamination method was the standardN-Acetyl-L-Cysteine-2% sodium hydroxide (NALC-NaOH) procedure (Kent etal., 1985. Public health mycobacteriology: a guide for level IIIlaboratory. US Dept. of Health and Human Services, Centers for DiseaseControl, Atlanta, Ga.). NALC acts as a mucolytic agent to ensureliquefaction of the specimen and sodium hydroxide is a decontaminatingagent. Smear intensity was determined based on the usual clinicalclassification of mycobacteria culture where “1+” means a low positiveand “4+” means a high positive.

The results obtained for 12 specimens are summarized in Table 5. Theresults show the smear intensity for each specimen (column 2) comparedto the probe detection results obtained with the homogeneous detectionassay with a labeled probe (single assay RLU results, column 3) and theresults obtained following hybridization to the DNA probe array (column4, BC %, and column 5, signal intensity).

TABLE 5 DNA Probe Labeled Probe Array Result Result Signal Specimen No.Smear Intensity RLU BC % Intensity Sediment 1 3+ 6.37 × 10⁶ 97.6 30,734Sediment 2 3+ 5.60 × 10⁶ 95.9 27,284 Sediment 3 3+ 1.09 × 10⁵ 94.3 1,046Sediment 4 4+ 2.36 × 10⁶ 99.2 12,702 Sediment 5 4+ 6.86 × 10⁵ 98.4 8,273Sediment 6 4+ 3.90 × 10⁶ 98.4 17,204 Sediment 7 4+ 6.02 × 10⁶ 99.2 8,619Sediment 8 3+ 4.867 × 10⁶  99.2 6,391 Sediment 9 3+ 2.99 × 10⁶ 98.45,178 Sediment 10 3+ 1.98 × 10⁶ 99.2 3,196 Sediment 11 2+ 2.08 × 10⁶96.7 820 Sediment 12 2+ 3.50 × 10⁶ 98.4 2,615The results shown in Table 5 demonstrate the efficiency of amplificationwith clinical specimens. For all sediments tested, the labeled probedetection results (RLU) were all positive compared to a negative control(not shown), and the DNA probe array detection results were similarlypositive. In the probe array analysis, all of the tested sediments weredetected as wild type M. Tuberculosis.

EXAMPLE 5 PCR Amplification and Detection of Amplicons

This example shows the sensitivity of primers of the present inventionwhen used in another amplification method, the polymerase chain reaction(PCR). The amplified DNA was detected on a solid support having an arrayof immobilized probes (i.e., GENECHIP™). For target preparation, wildtype M. Tuberculosis (ATCC No. 27177) was grown in vitro using standardmicrobiology methods. Bacterial stock suspensions were made in water andadjusted to a concentration of about 6×10⁸ bacteria per ml. Successivedilutions in water were made to produce a concentration of 10⁴ bacteriaper μl (equivalent to 10⁴ copies of bacterial DNA per μl) which werethen inactivated by heating for 15 min at 95° C.

PCR amplification was carried out in a plastic tube using a thermostableDNA polymerase isolated from Thermus aquaticus (FAST START™ Taq DNApolymerase, Roche Molecular Biochemicals). Briefly, the amplificationmixture (50 μl final volume) contained 5 μl of 10× amp buffer, 0.4 μl ofdNTP mix (25 mM each of ATP, CTP, UTP and GTP), 1.5 μl each of primersof SEQ ID NO:2 and SEQ ID NO:3 (10 μM), 0.4 μl of DNA polymerase (2U), 5μl of target (or water for the negative control). Thermal cycling wasperformed using an automated thermal cycler (PERKIN-ELMER 9600™) with aninitial denaturation step at 95° C. for 4 min, followed by 35 cycleseach consisting of 95° C. for 30 sec, 50° C. for 30 sec and 72° C. for45 sec, and a final cycle of 72° C. for 7 min.

Following PCR amplification, the amplification products were analyzed byagarose gel electrophoresis and stained with ethidium bromide, to detectthe presence or absence of a 168 nt band of amplified DNA. No band wasvisible on the gel for the negative control (i.e., without target DNA).A DNA band was seen when a reaction included 10⁴ or more copies oftarget.

Next, amplification products were detected on a DNA probe array(GENECHIP™), substantially as described previously (Troesch et al.,1999, J Clin. Microbiol. 37(1):49-55). Promoter-tagged PCR ampliconswere used to generate single-stranded RNA targets by in vitrotranscription reactions (20 μl) that each contained about 50 ng of PCRproduct, 20 U of T7 RNA polymerase (Promega), 40 mM Tris acetate (pH8.1), 100 mM Mg(acetate)₂, 10 mM dithiothreitol, 1.25 mM each of ATP,CTP, UTP and GTP, and were incubated at 37° C. for 1 hr.

The RNA was fluorescently labeled substantially as described (PCT No. WO01/44507) and the labeled RNA was hybridized to the probe array andanalyzed (Troesch et al., supra). For hybridization, 5 μl the labeledRNA was diluted in 700 μl of hybridization buffer (0.90 M NaCl, 60 mMNaH₂PO₄, 6 mM EDTA, pH 7.4, and 0.05% (v/v) TRITON® X-100), applied tothe probe array and incubated at 45° C. for 30 min. Then the probe arraywas washed twice in 3×SSPE (0.45 M NaCl, 30 mM NaH₂PO₄, 3 mM EDTA, pH7.4) and 0.005% (v/v) TRITON® X-100 at 30° C. and the fluorescent signalbound to the array was detected. The detected signal intensities (mean,median and maximum RFU), nucleotide % base calling and sequencedeterminations were generated by using the system algorithm (GENECHIP™software, Affymetrix). A candidate selection index was determined by thepercentage of homology between the experimentally derived sequence andreference sequences present on the array. The results obtained from thisexperiment showed that for 104 copies of target the sequence determinedon the probe array was that of wild type M. Tuberculosis, based on 91.9%base calling and a signal intensity of 1312 RFU, compared to thebackground signal of 101 RFU.

EXAMPLE 6 Amplification of rpoB (+) Strand Target

This example shows that the assay can amplify and detect amplicons madefrom the rpoB region but from the (+) DNA strand for comparison to the(−) DNA strand amplification described in Example 1. Using substantiallythe same amplification conditions as in Example 1, the amplificationoligonucleotides used in the (+) strand amplification reaction were: SEQID NO:10 (AATTTAATACGACTCACTATAGGGAGAACGCTCACGTGACAGAC) as a promoterprimer and SEQ ID NO: 11 (GGTCGCCGCGATCAAG) as a primer. The targetsequences for this assay were purified gDNA extracted from a lysedbacterial culture of M. Tuberculosis and provided at 5×10³ copies peramplification reaction or a crude lysate of sonicated M. Tuberculosiscells grown in broth culture, provided at about 5×10³ copies peramplification reaction. As a negative control, an equal volume of watercontaining no M. Tuberculosis DNA was substituted for the DNA-containingsamples in a separate amplification reaction that was processed as forthe positive samples. Amplification was assessed based on the detectedchemiluminescence (RLU) after a homogeneous detection assay performedsubstantially as described above but using the AE-labeled detectionprobe of SEQ ID NO:12 (CATGAATTGGCTCAGCTG). For both the purified gDNAand the crude lysate samples, positive signals were detected. For tworeplicate assays with purified gDNA targets the average signal detectedwas 5.66×10⁶ RLU, and for five replicate assays with crude lysate DNAtargets the average signal detected was 5.25×10⁶ RLU. The negativecontrol (two replicate assays) gave 6.15×10² RLU. These results showthat the assay can specifically detect rpoB sequences independent of thestrand of M. Tuberculosis DNA that is amplified.

1. A method of detecting rpoB sequences of Mycobacterium tuberculosispresent in a biological sample, comprising the steps of: providing abiological sample containing nucleic acid from M. tuberculosiscomprising a rpoB sequence; amplifying the rpoB sequence in an in vitronucleic acid amplification reaction mixture comprising at least onepolymerase activity, and a first primer consisting of SEQ ID NO: 10 anda second primer consisting of SEQ ID NO: 11 to produce amplified nucleicacid containing a rpoB sequence; optionally fragmenting the amplifiednucleic acid; hybridizing the amplified nucleic acid to at least onedetection probe consisting of SEQ ID NO:12 that hybridizes specificallyto M. tuberculosis sequences; and detecting the amplified nucleic acidhybridized to at least one detection probe by detecting a labelassociated with the amplified nucleic acid, thereby detecting rpoBseguences of Mycobacterium tuberculosis present in the biologicalsample.
 2. The method of claim 1, before the amplifying step, furthercomprising the steps of: adding to the biological sample a combinationof first and second capture oligomers consisting of SEQ ID NO: 5 and SEQID NO: 9 wherein the first and second capture oligomers specificallyhybridize to a M. tuberculosis sequence specific for each captureoliciomer, and an immobilized nucleic acid that hybridizes to a 3′ tailsequence of the first and second capture oligomers; hybridizing thenucleic acid from M. tuberculosis, the capture oligomers, and theimmobilized nucleic acid to produce a hybridization complex comprisingthe nucleic acid from M. tuberculosis at least one of the first andsecond capture oligomers, and the immobilized nucleic acid; andseparating the hybridization complex from other components of thebiological sample.
 3. The method of claim 1, wherein the amplifying stepuses a helper oligomer consisting of SEQ ID NO:
 1. 4. The method ofclaim 1, wherein amplifying comprises transcription-mediatedamplification.
 5. The method of claim 1, wherein amplifying comprisespolymerase chain reaction (PCR) amplification.
 6. The method of claim 1,wherein the method includes chemically fragmenting the amplified nucleicacid and fluorescently labeling fragments of the amplified nucleic acid.7. A composition for detecting a rpoB sequence of M. tuberculosis,comprising an oligonucleotide consisting of SEQ ID NO: 10 and anoligonucleotide consisting of SEQ ID NO: 11, and optionally at least onedetection probe consisting of SEQ ID NO:
 12. 8. The composition of claim7, further comprising an oligonucleotide consisting of SEQ ID NO:
 1. 9.The composition of claim 7, further comprising a combination of a firstoligonucleotide consisting of SEQ ID NO: 5 and a second oligonucleotideconsisting of SEQ ID NO:
 9. 10. A kit comprising at least anoligonucleotide consisting of SEQ ID NO: 10 and an oligonucleotideconsisting of SEQ ID NO: 11, and at least one oligonucleotide selectedfrom the group consisting of an oligonucleotide consisting of SEQ ID NO:1 and an oligonucleotide consisting of SEQ ID NO:
 12. 11. The kit ofclaim 10, comprising the oligonucleotide consisting of SEQ ID NO:
 1. 12.The kit of claim 10, comprising the oligonucleotide consisting of SEQ IDNO:
 12. 13. The kit of claim 10, further comprising a combination of anoligonucleotide consisting of SEQ ID NO: 5 and an oligonucleotideconsisting of SEQ ID NO:
 9. 14. The kit of claim 10, further comprisingan oligonucleotide consisting of SEQ ID NO:
 2. 15. The kit of claim 10,comprising the oligonucleotide consisting of SEQ ID NO: 1 and theoligonucleotide consisting of SEQ ID NO: 12.